This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The visual photoreceptor rhodopsin is a prototypical seven transmembrane (7-TM) helical G-protein coupled receptor (GPCR) that is responsible for light detection under dim light conditions in the scotopic visual system. The detailed structure of the binding site of the covalently attached 11-cis retinylidene chromophore has been determined, confirming data from functional characterization. However, the entry pathway in the opsin apoprotein for the hydrophobic ligand 11-cis retinal is unknown. Here we show that the primary ligand entry site is located in the transmembrane region between helices 5 and 6, suggesting a hydrophobic binding and unbinding pathway for the ligand through the bilayer interior. In addition to the primary entry site, a particular residue located in the second extracellular loop has a significant effect on chromophore regeneration rate, probably through an indirect effect on the helix 5-helix 6 pathway. Multi-nanosecond free energy calculations of ligand binding in a membrane model of rhodopsin were performed based on reversible MD simulations using umbrella potentials. The weighted histogram analysis method (WHAM) was employed to eliminate the effects of the biasing umbrella potentials and to recover potential of mean force (PMF) free energy profiles. The results are compared with irreversible steering molecular dynamics (SMD) simulations applying the Jarzynski nonequilibrium equality, and with multi-configurational thermodynamic integration (MCTI). In the previous application period, we were able to extend the timescale of the SMD trajectories from 4x2 nanoseconds to 6x20+100 nanoseconds. Comparing the obtained structural intermediates of the proposed dissociation pathway, we found that we need long trajectories to avoid artifacts, such as protein deformations that lead to 'flooding'of water molecules into internal cavities. In repeated simulations, we found that two alternative pathways in the helix 5/helix 6 interface are taken by the ligand. The 'upper'pathway leads to the lipid/water interface, while the 'lower'pathway leads to the bilayer center. These two pathways are also predominant in a 'brute force'search methodology, which identifies an additional 'weak point'of the binding pocket in the helix 1/helix 2 interface. It will be very important to repeat the 100 nanosecond simulations several times, and to explore the energetics of additional pathway between helices 1 and 2. In addition to tryptophan fluorescence resonance energy transfer (FRET) experiments on site-directed mutant and wild type receptors that show three orders of magnitude changes of retinal binding kinetics depending on side chains in the entry pathway, we obtained during the previous application period the missing thermodynamic data from dissociation kinetics and isothermal titration calorimetry. The results suggest high energy barriers for ligand entry and exit. Hydrogen bond donor side chains along the pathway lead to kinetic trapping of retinal. Models of members of the family of visual pigments based on sequence homology suggest that these findings could explain the rapid recovery of the receptors following bleaching in the photopic system for bright-light and color vision. The implications of these finding are reaching far beyond the visual system;GPCRs are an important group of drug targets. The only high resolution structure of a GPCR publicly available is that of the visual photoreceptor rhodopsin. It is therefore obvious to evaluate the ligand binding mechanism in this system as a potential benchmark for the development of computational drug design methods. In this respect, our approach will provide the first complete view of the ligand binding mechanism in a 7-TM receptor based on both experiments and atomistic simulations.